Human immunodeficiency virus type 1 (HIV-1) is the major cause of acquired immunodeficiency syndrome in humans. Rhesus monkey TRIM5alpha (rhTRIM5a) is a potent intrinsic anti-retroviral factor which targets incoming HIV-1 capsid and promotes its premature disassembly. rhTRIM5a comprises an RBCC domain and a B30.2(SPRY) domain, and sequences in the B30.2(SPRY) domain dictate the potency and specificity of the restriction. Since rhTRIM5a recognizes proteolytically processed capsids in incoming virions, it was believed that rhTRIM5a blocks only the early phase of the HIV-1 life cycle. Contrary to this general consensus, we have recently discovered that rhTRIM5a, but not its human orthologue (huTRIM5a), potently blocks HIV-1 production by reducing the levels of Gag proteins in producer cells. Unexpectedly, the rhTRIM5a RBCC domain, but not the B30.2(SPRY) domain, plays a pivotal role in the block of HIV-1 production. Moreover, when HIV-1 Gag is over-expressed, encapsidation of rhTRIM5a is observed in viral-like particles made with HIV-1 Gag polyproteins, suggesting the interaction between rhTRIM5a and HIV-1 Gag polyprotein during viral production. Our long-term goal is to develop novel intervention strategies to block HIV-1 replication based on understanding of anti-HIV-1 activities of host restriction factors. The overall goal of this application is to understand the underlying molecular mechanisms of the rhTRIM5a-mediated block of HIV-1 production; a clear understanding is essential for any rational attempt to block virus production by small molecules. Our central hypothesis is that rhTRIM5a interacts with HIV-1 Gag polyprotein before viral assembly and promotes rapid degradation of Gag, thereby blocking HIV-1 production. Specific aims are; 1) to characterize the nature and biological relevance of the rhTRIM5a-mediated block on lenti/retroviral production, 2) to determine the essential biological properties of rhTRIM5a for the block of HIV-1 production, and 3) to determine how rhTRIM5a reduces the levels of HIV-1 Gag in the producer cells. Accomplishing the specific aims outlined in this proposal should lead to considerable insights into how rhTRIM5a blocks HIV-1 production and it will also reveal potential targets for HIV-1 therapy. For instance, it may be possible to develop molecular strategies which transform huTRIM5a into an effective inhibitor of HIV-1 production.